1. Technical Field
The present invention relates to a fluorescent reagent using semiconductor nanoparticles, and a fluorescence determination method using semiconductor nanoparticles.
2. Background Art
Semiconductor nanoparticles of a grain size of 10 nm or less are located in the transition region between bulk semiconductor crystals and molecules. Their physicochemical properties are therefore different from both bulk semiconductor crystals and molecules. In this region, the energy gap of a semiconductor nanoparticle increases as its grain size decreases, due to the occurrence of a quantum-size effect. In addition, the degeneracy of the energy band that is observed in bulk semiconductors is removed and the orbits are dispersed. As a result, a lower-end of the conduction band is shifted to the negative side and an upper-end of the valence band is shifted to the positive side.
Semiconductor nanoparticles can be easily prepared by dissolving equimolar amounts of precursors of Cd and X (X being S, Se or Te). This is also true for their manufacture using CdSe, ZnS, ZnSe, HgS, HgSe, PbS, or PbSe, for example.
The reason semiconductor nanoparticles are attracting attention is that since semiconductor nanoparticles are characterized by emitting strong fluorescence with a narrow full width at half maximum, the creation of various colors of fluorescence is possible. Thus, it is considered that future applicable fields are almost unlimited. However, semiconductor nanoparticles obtained by the above method exhibit a wide grain-size distribution and therefore cannot provide the full advantage of the properties of semiconductor nanoparticles.
Therefore, attempts have been made to attain a monodisperse distribution by using chemical techniques to precisely separate the semiconductor nanoparticles having a wide grain-size distribution immediately after preparation into individual grain sizes and extract only those semiconductor nanoparticles of a particular grain size. The attempts to attain a monodisperse distribution of grain size that have been reported so far include an electrophoresis separation method that utilizes variation in the surface charge of nanoparticles depending on the grain size, an exclusion chromatography that takes advantage of differences in retention time due to different grain sizes, a size-selective precipitation method utilizing differences in dispersibility into an organic solvent due to different grain sizes, and a size-selective optical etching method that takes advantage of the fact that a metal chalcogenide semiconductor is oxidatively dissolved when irradiated by light in the presence of dissolved oxygen.
Semiconductor nanoparticles obtained by these methods exhibit a spectrum having a peak with a relatively narrow full width at half maximum (FWHM). Thus, by controlling the grain size of semiconductor nanoparticles, various reagents exhibiting a spectrum having narrow FWHMs can be prepared. This enables multicolor analyses for the detection and imaging of biopolymers. Further, semiconductor nanoparticles have greater durability compared with commonly used organic dyes, and they are almost free from fading.
Also, in addition to band gap fluorescence exhibited by the inner part of semiconductor nanoparticles, semiconductor nanoparticles emit defect fluorescence that is completely different from fluorescence arising from an energy level existing in the forbidden band of energy levels inside semiconductor nanoparticles.
The energy level that emits the defect fluorescence is presumably derived from the presence of a defect level mainly on the surface site of semiconductor nanoparticles and is considered to inhibit the properties of semiconductor nanoparticles exhibiting a spectrum with a narrow FWHM, and thus this has been a problem to be solved. Further, as described later using FIG. 2, when semiconductor nanoparticles are prepared using a size-selective optical etching method or the like, the phenomena may be observed where defect fluorescence generated from the defect level is more strongly emitted than inherent fluorescence generated from the band gap inside the semiconductor nanoparticles. In the present invention, fluorescence that is exhibited due to the presence of a defect level mainly on the surface site of semiconductor nanoparticles is called “defect fluorescence.”
As a typical solution method to overcome the effect of this defect fluorescence, a method has been attempted which carries out multi-layering on the semiconductor material for the particle by coating the core with a semiconductor material having a broader band gap than the semiconductor material for the core, and inorganic and organic materials, and suppresses the defect fluorescence. Experiments by this method have been carried out with various materials. However, because the preparation of semiconductor nanoparticles by this method requires the safety of reagents and a reaction at relatively higher temperatures, the method can be hardly said to be industrially preferable. When semiconductor nanoparticles are not multi-layered, the phenomena may be observed where florescence generated from the defect level thereof is stronger than inherent fluorescence generated from the band gap inside semiconductor nanoparticles.
Therefore, there has been a need to solve the problem of defect fluorescence inhibiting measurement of the inherent fluorescence of semiconductor nanoparticles.